The great greenbriers gall mystery resolved? New species of Aprostocetus Westwood (Hymenoptera, Eulophidae) gall inducer and two new parasitoids (Hymenoptera, Eurytomidae) associated with Smilax L. in southern Florida, USA

Aprostocetus smilax Gates & Zhang, sp. nov., is described from stem and leaf galls on Smilax havanensis Jacq. in southern Florida, USA. It is the third species of Aprostocetus Westwood known to induce plant galls. Two parasitoids of A. smilax are also described: Phylloxeroxenus smilax Gates & Zhang sp. nov. and Sycophila smilax Gates & Zhang, sp. nov. We conclude that A. smilax is the true gall inducer on Smilax L., and thus the host records of Diastrophus smilacis Ashmead and its inquiline Periclistus smilacis Ashmead, both from Smilax, are erroneous.


Introduction
Gall induction in Chalcidoidea was summarized by La  wherein he noted its occurrence in six families, representing at least 15 independent origins. Within Eulophidae multilocular galls and notes were made about the contents of each locule in terms of its condition and occupant prior to each occupant being assigned a code and preserved in 80% ethanol. We noted six ectoparasitoid specimens. Pertinent taxon-specific notes are included in results below.

Imaging
Ethanol-preserved specimens were dehydrated through increasing concentrations of ethanol, and transferred to hexamethyldisilazane (HMDS) (Heraty and Hawks 1998) before point-mounting. MWG identified parasitoids using a Leica M205C stereomicroscope with 10X oculars and a Leica LED ring light source for point-mounted specimen observation. We took scanning electron microscope (SEM) images with a Hitachi TM3000 (Tungsten source). Body parts of disarticulated specimens were adhered to a 12.7 × 3.2 mm Leica/Cambridge aluminum SEM stub by a carbon adhesive tab (Electron Microscopy Sciences, #77825-12). Stub-mounted specimens were sputter coated with gold-palladium using a Cressington Scientific 108 Auto from multiple angles to ensure complete coverage (~20-30 nm coating). Habitus images were obtained using a Visionary Digital imaging system. The system consists of a Canon EOS 5D Mark II digital SLR camera with a 65 mm macro lens. A Dynalite MP8 power pack and lights provided illumination. Image capture software was Visionary Digital's proprietary application with images saved as TIF with the RAW conversion occurring in Canon Digital Photo Professional software. Image stacks were montaged with Helicon Focus 6.2.2. Image editing was done in Adobe Photoshop and plate layout in Adobe Illustrator. The painting was made from pinned and live insect specimens, plant herbarium sheets and photographs. Additional structural details of the insects were obtained from SEM photographs. The final image was painted using Adobe Photoshop.
We used several species keys to determine whether our material belonged to any described species (Balduf 1932;Graham 1987) with details below under each specific treatment. Where possible, all species identifications were corroborated by comparison with authoritatively identified specimens in the Smithsonian National Museum of Natural History.

Molecular protocol
Specimens were extracted, amplified, and sequenced at USDA Beltsville Agricultural Research Center (BARC) using the DNeasyTM Tissue Kit protocol (Qiagen, Valencia, CA, USA). Specimens were digested for circa three hours using 20 μL of 20 mg/ mL Proteinase K at 55 °C. The DNA was resuspended with 150 μL of Qiagen elution buffer. Fragments of mtDNA COI (620 bp) were amplified using LCO1490 5'-GGT-CAACAAATCATAAAGATATTGG-3' and HCO2198 5'-TAAACTTCAGGGT-GACCAAAAAATCA-3' (Folmer et al. 1994). Amplifications for rDNA 28S (820 bp) used 28S_D1F 5'-ACCCGCTGAATTTAAGCATAT-3' (Harry et al. 1996) and 28S_ D2R 5'-TTGGTCCGTGTTTCAAGACGGG-3' (Campbell et al. 1994). All PCRs were performed using approximately 2 μL DNA extract, 1.25 μL 10× Buffer, 1 μL dNTP, 1 μL of each primer, 1 unit of Taq DNA polymerase (TaKaRa Bio, Mountain View, CA, USA), and purified water for a final volume of 25 μL. Amplicons of COI were generated with an initial denaturation of 1 min at 95 °C, followed by 35 cycles of 95 °C for 15 s, 49 °C for 15 s and 72 °C for 45 s, and a final elongation period of 4 min at 72 °C. The thermocycler setting for 28S is similar to COI, with the exception of annealing temperature being at 55 °C. Sequencing was conducted using a ABI 3730xl DNA sequencer following manufacturer's instructions. Contigs were assembled and edited using Sequencher version 4.5 (Gene Codes). DNA sequences were then compared with available sequences in the Barcode of Life Database (BOLD, Ratnasingham and Hebert 2007) and the Basic Local Alignment Search Tool (BLAST) for nucleotides in GenBank. All sequences are uploaded onto GenBank (see Table 1).

Aprostocetus smilax
and speciose of the five Aprostocetus subgenera, often associated with insects inhabiting plant galls such as Diptera (Cecidomyiidae), Hymenoptera (Cynipoidea), Hemiptera (Coccoidea), Coleoptera, and eriophyid mites (La . Burks (1967) published a key to 13 North American species, which is dated, and a comparative diagnosis of all 58 species is beyond the scope of this paper. Nevertheless, this species keys to couplet 2 of Burks' key, and differs from the two species with coriaceous mesoscutum (A. coelioxydis Burks and A. granulatus Ashmead) which are both metallic blue/black in coloration. Recent phylogenomic study of Eulophidae has shown Aprostocetus to be paraphyletic (Rasplus et al. 2020), and some of these subgenera might be elevated to genus level in the future. Description. Female. Body length 1.7 mm (Fig. 2).
Male. 1.1 mm. Color and sculpture as described for female (Fig. 3). Antennae with setae >1.5× as long as width of segment (Fig. 15). Gt7 curves up to form genital opening (Figs 16, 17), with a pair of long and three pairs of shorter cercal setae (Fig. 18).
Variation. Size ranges from 1.6-1.8 mm for females, and 1.1-1.2 mm for males. The number of setae on marginal vein ranges from 6-8.
Biology. It induces round galls on the stems of Smilax havanensis, often coalescing to form irregularly rounded, polythalamous swellings. Individual galls can also be found on the edge of leaves. Green when fresh and of a pithy structure (Figs 1 (Ashmead), which is suspected to be a parasitoid of the cecidomyiid inquiline within Phylloxera Boyer de Fonscolombe galls on hickory (Carya Nutt.) (Ashmead 1881). The lower face is strigose and the ventral half of the body is yellow in P. smilax, while in P. phylloxerae the lower face is striate and the body is completely black. There are at least 50 undescribed species in at least three species groups for the Neotropical region that exhibit a range of variation in diagnostic generic characters such as the propodeum in lateral view forming a 90° angle with mesosoma; long/short petiole and resultant effect on striate part of S1 (Fig. 30), with the striae on S1 being a reliable diagnostic though expressed to varying degrees; and lower face with/without striae (Gates, unpublished data Description. Female. Body length 1.88 mm (Fig. 19). Color. Orange-yellow; antennal segments light brown; edges of ocelli, scutellum, metasoma mediodorsally with black band, eyes pinkish red (Fig. 19).
Male. 1.51 mm. Color and sculpture as described for female (Fig. 20). Antennal with funicular segments pedicellate, each with 2 or more rows of erect setae and about 1.5× as long as width of segment. Four funicular segments and a trisegmented clava (Fig. 32). Gastral petiole in lateral view cylindrical with projecting lateral teeth and mediodorsal prong (Fig. 34), in dorsal view length about 2.5× as long as greatest width, 1.6× as long as the length to metacoxa; evenly reticulate dorsally and ventrally (Fig. 33), smooth laterally.
Variation. Size ranges from 1.76-1.91 mm for females, and 1.45-1.52 mm for males. The coloration on the body can range from almost completely yellow, to mostly black on the dorsolateral surfaces, particularly in males.
Biology. Associated with galls of Aprostocetus smilax, likely a parasitoid of the gall inducer.
Forewing. Dark brown band on the wing about the same width as pterostigma and does not reach uncus, faint, reaching about ½ down the wing width, 8 submarginal setae, 3 on parastigma, 1 in basal cell, surrounded by basal and costal setal lines. Pterostigma covering marginal, postmarginal, and stigmal vein.
Variation. Body ranges 1.7-1.8 mm for females, 1.65-1.88 mm for males. The wing band can range from very faint, mesosoma and metasoma dorsally can be yellow or with a tinge of black.
Biology. Associated with galls of Aprostocetus smilax, likely a parasitoid of the gall inducer.

Molecular analyses
A total of 55 individuals had both or at least one of the two genes sequenced. BLAST and BOLD search results confirmed the family and sometimes genus level identification, but did not return any hits at the species level. This Smilax gall contains 3 different families of chalcidoids: the majority of the gall inhabitants consisted of the suspected gall inducer Aprostocetus smilax (n = 40), and two eurytomid parasitoids Phylloxeroxenus smilax (n = 7) and Sycophila smilax (n = 6) (Fig. 63). Specimen G0042 was identified as an unknown tetrastichine eulophid that was destructively sampled, while G0052 was identified as Brasema Cameron (Eupelmidae) (Fig. 63). This Brasema specimen was never reared as an adult from this system, we noted it encircling another larva, presumably the gall inducer, and characterized by large mandibles and erect setae.

Validity of Cynipidae associated with Smilax
As the result of this study, the validity of Diastrophus smilacis (Figs 54, 55) inducing galls on Smilax was also investigated. The resulting fieldwork revealed Aprostocetus smilax is the true gall inducer in Florida, after some 400 galls never yielded any cynipids. Further, dissections of the galls from which the type specimen of D. smilacis was reared from (collected in Illinois) revealed vascular tissue patterns consistent with dicots and not monocots (Fig. 56). As no additional material of D. smilacis has been found since its original description, despite extensive searches in Illinois (Zhiwei Liu, pers. comm.) and other parts of North America (Weld 1959), we can safely conclude Smilax is not the host of Diastrophus smilacis.
Working with the type material of both D. smilacis and Periclistus smilacis Ashmead (Figs 59, 62, the putative inquilline of D. smilacis) revealed additional curiosities that require mentioning here. Ashmead (1896a) reports specimens of D. smilacis were apparently sent to C.V. Riley from Chicago, Illinois (Figs 57,58), and that Ashmead intended to describe them but the publication of the manuscript was delayed due to C.V. Riley's untimely death. Ergo, time passed, and in the same year (1896), in two different publications, we find the descriptions of D. smilacis (Ashmead 1896a) and P. smilacis (Ashmead 1896b). While this is not an entirely foreign set of circumstances, the specimens referred to in these two publications are quite confusing.
Ashmead (1896a) reports 13 specimens (females) for the description of D. smilacis, but the taxon is only known from the type specimen in the USNM and there is no record of additional specimens being loaned out; one cotype of this taxon is in AMNH, for a total of two specimens. The gall with the same type specimen number as the holotype wasp in the USNM (No. 3096, Fig. 57) has the label '86x' affixed to the pin, and it is mentioned in Ashmead (1896a) that a gall was collected for this species in Florida, but no wasp. Hence, Illinois is the origin of all material associated with D. smilacis. Ashmead (1896b) describes P. smilacis from 17 specimens and goes on to say the collection data for 13 specimens (same number as D. smilacis, above) is labeled 'No. 864, reared April 28, 1871 and four numbered 1010, reared February 4, 1884, from Diastrophus smilacis'. However, the type specimen of P. smilacis (Fig. 61) in the USNM has label data consistent with the label data of D. smilacis, suggesting Ashmead (1896b) erroneously read '86x' as '864' and that the same gall that yielded the type  specimens of D. smilacis yielded the type specimens for P. smilacis; there is no date on the '86x' specimens and it is not clear how the collection date in Ashmead (1896b) was obtained. The four specimens labeled '1010' cannot be located and are presumed either lost or in another, unreported museum.
Adding to this confusing picture is that it appears A. Ritchie intended to include P. smilacis in his dissertation work on Periclistus in 1984, and even went so far as to designate a lectotype for this species (Fig. 61). The series of specimens seen in Fig. 60 is the source of the specimen that Ritchie intended as the lectotype, making the total number of specimens for P. smilacis, in the USNM, 11 specimens. When we consider D. smilacis is represented by two specimens (holotype in USNM, cotype in AMNH), we have a grand total of 13 specimens. Our conclusion from all of this is that the original 13 specimens mentioned in Ashmead (1896a) for D. smilacis turned out to be a mixture of gall inducer and inquilline, and further, the host plant for this gall was mis-identified in the field as Smilax rotundifolia L. The US Forest Service Fire Effects Information System indicates S. rotundifolia and Rubus spp. co-occur in old fields throughout the range of Smilax and it is possible that the galls of Diastrophus smilacis are actually collected from a Rubus, and the two host plants were confused when the original collection was made.
The original collections made in Florida in 2010 that led to the chalcidoids described herein were also focused on the (now) erroneous records of D. smilacis on Smilax havanensis mentioned in Beutenmüller (1909) collected around Miami by Dr. E. Bessey. When looking closely at the D. smilacis gall figured in Beutenmüller (1909), it is clear that gall matches exactly what was collected in this project and illustrated in Fig. 1. No gall material from S. havanensis is in the cynipid gall collection, and indeed, there are no galls in this collection that look like the one figured in Beutenmüller (1909). As no cynipids apparently emerged from the Miami gall reported and figured in Beutenmüller (1909), we consider this an erroneous host record as well.
Lastly, the USNM has a specimen labeled as lectotype for Periclistus smilacis, yet this taxon lacks a published lectotype designation. We presume the team of Ritchie and Shorthouse, whose names appear on the purported lectotype labels, planned to publish these designations (as mentioned above), but were not able to. In order to stabilize the name of Periclistus smilacis, we hereby designate USNMENT00802336, type number 3287, as lectotype of this taxon, deposited in the USNM (Figs 59, 61, 62).

Conclusion
Here we describe the new eulophid species Aprostocetus smilax, the second recorded case of gall induction by Aprostocetus in North America. This new species is the true gall inducer on Smilax, and previous records of cynipid species Diastrophus smilacis and the inquiline Periclistus smilacis associated with this host plant are erroneous. Additionally, we described two eurytomid parasitoids associated with this Smilax gall. The distribution of all three new species is on the southern tip of mainland USA, but it is likely that they are also found in the Caribbean region in which the host plant S. havanensis is found (Ferrufino-Acosta 2014). A comprehensive taxonomic revision of these incredibly diverse but understudied minute wasps will undoubtedly reveal additional ecological associations and new species. staff from Miami-Dade County (C. Rodriguez and T. Smith) provided specimens and access to Rockdale Pineland for gall collecting. We would also like to thank Gary Oullette for performing all DNA extractions and amplifications, Taina Litwak for the illustration and image editing, and Zhiwei Liu for discussion on the validity of Diastrophus smilacis. Finally, we would like to thank Paul Hanson and an anonymous reviewer that have provided comments that improved the manuscript. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. USDA is an equal opportunity provider and employer.